Neoplastic development of Syrian hamster embryo (SHE) cells is a multistep process. However, the number of steps and the genes or changes involved are unknown. We have compared the susceptibilities of normal and carcinogen-induced preneoplastic SHE cells to transformation following transfection with plasmids of genomic clones of oncogenic viruses: polyoma virus, Harvey murine sarcoma virus (v-Ha-ras), and MC29 virus (pSVv-myc). Normal SHE cells transfected with polyoma virus DNA formed tumors within 3-4 weeks when injected into nude mice. In contrast, SHE cells treated with v-Ha-ras DNA remained nontumorigenic. SHE cells transfected with v-Ha-ras DNA plus v-myc DNA formed tumors with short latency periods. These results suggest that multiple changes or activated oncogenes are required for the neoplastic transformation. To determine whether activation of ras plus myc was sufficient for tumorigenicity, we performed cytogenetic analyses of tumors formed following transfection of Syrian hamster embryo cells with v-Ha-ras plus v-myc DNAs or polyoma DNA alone. Whereas polyoma-induced, tumor-derived cells were diploid, tumors induced by v-Ha-ras plus v-myc oncogenes were monoclonal and had a nonrandom chromosome change, monosomy of chromosome 15. Thus, an additional change, loss of chromosome 15, is required or advantageous for tumorigenicity induced by v-Ha-ras plus v-myc oncogenes. These results suggest that neoplastic progression of normal, diploid cells requires more than two steps under certain conditions. To determine if normal cellular factors or genes can regulate the phenotypic expression of tumorigenicity and/or oncogenes, cell-cell hybrids between chemically transformed SHE cells and either normal or preneoplastic SHE cells were prepared. Anchorage independence and tumorigenicity were suppressed in hybrids between tumorigenic and normal cells and in hybrids between tumorigenic and most but not all preneoplastic cells. This suggests that this suppressive ability may be lost during neoplastic progression and represents one step in this process.